Target (protein/gene name): GES-5 carbapenemase (blaGES-5) NCBI Gene # or RefSeq#:9389494 Protein ID (NP or XP #) or Wolbachia#: (none available on PubChem) Organism (including strain):Klebsiella pneumoniae Etiologic Risk Group (see link below): RG2
Background/Disease Information (sort of like the Intro to your Mini Research Write up): Klebsiella pneumoniae causes a type of pneumonia that is associated with destructive changes in the lungs. It has a rapid onset and often-fatal outcome despite early and appropriate antimicrobial treatment. Patients with this type of penumonia typically experience an acute onset of high fever and chills, flulike symptoms, and productive cough with abundant, thick, blood-tinged sputum (1). Klebsiella bacteria can be spread from person-to-person contact and less commonly by contamination of the environment (not spread through air). From contact, Klebsiella bacteria may enter the respiratory tract or bloodstream. Patients whose care requires devices like ventilators, intravenous catheters and patients who take long courses of certain antibiotics are most at risk (2) Klebsiella bacteria have developed antimicrobial resistance, most recently to the class of antibiotics known as carbapenems (2). This is because Klebsiella pneumoniae are beginning to make carbapenemases, enzymes that break down carbapenems. making them ineffective. One report cites that they carbapenemases can contribute to death in up to 50% of patients who become infected. Carbapenemase-producing K. pneumoniae was very uncommon in the United States before 1992 and the first report on its prevalence in the US was not reported until 2001 (3). Carbapenemase-producing bacteria have also emerged in multiple species of gram-negative bacteria across the world (4).
Link to TDR Targets page (if present): None available Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):http://www.ncbi.nlm.nih.gov/gene/9389494 Essentiality of this protein: Yes "Acquired resistance to broad-spectrum β-lactams is increasingly observed in P. aeruginosa. Currently, PER-, VEB-, and GES-type enzymes are the most frequently observed extended-spectrum β-lactamases (ESBLs) identified in Pseudomonas spp. Therefore, carbapenems are considered crucial for treating many P. aeruginosa-associated infections." Article: Rapid Detection of Carbapenemase-Producing Pseudomonas spp. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486216/) Complex of proteins:No but commonly produced as a dimer (can still function as a monomer) Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
"The 28-day mortality was 13.3% in the combination therapy group compared with 57.8% in the monotherapy group (P = 0.01). The most commonly used combinations were colistin-polymyxin B or tigecycline combined with a carbapenem … The use of combination therapy for definitive therapy appears to be associated with improved survival in bacteremia due to KPC-producing K. pneumoniae." Article: Treatment Outcome of Bacteremia Due to KPC-Producing Klebsiella pneumoniae: Superiority of Combination Antimicrobial Regimens (http://aac.asm.org/content/56/4/2108.abstract)
Structure Available (PDB or Homology model) PDB#: 4H8R Pairwise alignment of your BLASTP search in NCBI against the PDB Query Coverage: 26-129 Max % Identities: 33/104 (32%) % Positives: 43/104 (41%) Chain used for homology: Chain A
Current Inhibitors: dCHEMBL777, TAZOBACTUM, and CHEMBL403 Expression Information (has it been expressed in bacterial cells): Yes Purification Method: β-Lactamase purification.Cultures of E. coli TOP10 harboring recombinant plasmid pTOPO-GES-14 were grown overnight at 37°C in 4 liters of TS broth containing amoxicillin (50 μg/ml) and kanamycin (30 μg/ml). β-Lactamase GES-14 was purified by ion-exchange chromatography. Briefly, the bacterial suspension was pelleted, resuspended in 40 ml of 100 mM sodium phosphate buffer (pH 7.0), sonicated, cleared by ultracentrifugation, and treated with DNase. The extract was then dialyzed against 20 mM bis-Tris {[bis(2-hydroxyethyl-imino]tris(hydroxymethyl)methane}(pH 6.5) and loaded onto a preequilibrated Q-Sepharose column with the same buffer. The resulting enzyme extract was recovered in the flowthrough and dialyzed against 20 mM Tris-HCl (pH 7.5) overnight at 4°C. This extract was then loaded onto a preequilibrated (20 mM Tris-HCl [pH 7.5]) Q-Sepharose column, and the proteins were eluted with a linear NaCl gradient (0 to 0.5 M). The β-lactamase activity was eluted with NaCl at a concentration of 200 mM in the same Tris-HCl buffer. Finally, fractions containing the highest β-lactamase activities were pooled and subsequently dialyzed overnight against 100 mM phosphate buffer (pH 7.0). The β-lactamase activity was determined qualitatively using nitrocefin hydrolysis (Oxoid, Dardilly, France). The protein content was measured using the Bio-Rad DC protein assay. Article: Carbapenem-Hydrolyzing GES-Type Extended-Spectrum β-Lactamase in Acinetobacter baumannii (http://aac.asm.org/content/55/1/349.long) Image of protein (PyMolwith features delineated and shown separately):
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MRFIHALLLAGIAHSAYASEKLTFKTDLEKLEREKAAQIGVAIVDPQGEIVAGHRMAQRFAMCSTFKFPLAALVFERIDSGTERGDRKLSYGPDMIVEWSPATERFLASGHMTVLEAA
QAAVQLSDNGATNLLLREIGGPAAMTQYFRKIGDSVSRLDRKEPEMSDNTPGDLRDTTTPIAMARTVAKVLYGGALTSTSTHTIERWLIGNQTGDATLRAGFPKDWVVGEKTGTCAN
GGRNDIGFFKAQERDYAVAVYTTAPKLSAVERDELVASVGQVITQLILSTDK
Length of your protein in Amino Acids: 287 residues Molecular Weight of your protein: 31184.5 kDa Molar Extinction coefficient of your protein at 280 nm wavelength:
25565
Abs 0.1% (=1 g/l) 0.820, assuming all pairs of Cys residues form cystines
25440
Abs 0.1% (=1 g/l) 0.816, assuming all Cys residues are reduced
TMpred graph Image:
CDS Gene Sequence (paste as text only): ATGTCACTGTATCGCCGTCTAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGCCACCGCGCTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGGA CTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCTTTCTTGCTGCC GCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGCAAAAATGCGCTGGTTCCGTGGTCACCCATCTCGGAAAAATATCTGACAACAGG CATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGTGATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGGCCGGGCTGACGGCCTTCATGCGC TCTATCGGCGATACCACGTTCCGTCTGGACCGCTGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGATGCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACA AAAACTGACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTTTGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGCGCGGCGGTGCCGGCAGA CTGGGCAGTCGGAGACAAAACCGGAACCTGCGGAGTGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCACTGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCGGG CGCCTAACAAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCG GCTAGACTCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAA GC% Content for gene: 61.5%
CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
Primer design results for 'tail' primers (this is just 2 sequences):
NCBI Gene # or RefSeq#: 9389494
Protein ID (NP or XP #) or Wolbachia#: (none available on PubChem)
Organism (including strain): Klebsiella pneumoniae
Etiologic Risk Group (see link below): RG2
Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Klebsiella pneumoniae causes a type of pneumonia that is associated with destructive changes in the lungs. It has a rapid onset and often-fatal outcome despite early and appropriate antimicrobial treatment. Patients with this type of penumonia typically experience an acute onset of high fever and chills, flulike symptoms, and productive cough with abundant, thick, blood-tinged sputum (1). Klebsiella bacteria can be spread from person-to-person contact and less commonly by contamination of the environment (not spread through air). From contact, Klebsiella bacteria may enter the respiratory tract or bloodstream. Patients whose care requires devices like ventilators, intravenous catheters and patients who take long courses of certain antibiotics are most at risk (2)
Klebsiella bacteria have developed antimicrobial resistance, most recently to the class of antibiotics known as carbapenems (2). This is because Klebsiella pneumoniae are beginning to make carbapenemases, enzymes that break down carbapenems. making them ineffective. One report cites that they carbapenemases can contribute to death in up to 50% of patients who become infected. Carbapenemase-producing K. pneumoniae was very uncommon in the United States before 1992 and the first report on its prevalence in the US was not reported until 2001 (3). Carbapenemase-producing bacteria have also emerged in multiple species of gram-negative bacteria across the world (4).
(1) http://emedicine.medscape.com/article/219907-clinical
(2) http://www.cdc.gov/HAI/organisms/klebsiella/klebsiella.html#a5
(3) http://www.cdc.gov/hai/organisms/cre/index.html
(4) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075864/
Link to TDR Targets page (if present): None available
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://www.ncbi.nlm.nih.gov/gene/9389494
Essentiality of this protein: Yes
"Acquired resistance to broad-spectrum β-lactams is increasingly observed in P. aeruginosa. Currently, PER-, VEB-, and GES-type enzymes are the most frequently observed extended-spectrum β-lactamases (ESBLs) identified in Pseudomonas spp. Therefore, carbapenems are considered crucial for treating many P. aeruginosa-associated infections."
Article: Rapid Detection of Carbapenemase-Producing Pseudomonas spp.
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486216/)
Complex of proteins:No but commonly produced as a dimer (can still function as a monomer)
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
"The 28-day mortality was 13.3% in the combination therapy group compared with 57.8% in the monotherapy group (P = 0.01). The most commonly used combinations were colistin-polymyxin B or tigecycline combined with a carbapenem … The use of combination therapy for definitive therapy appears to be associated with improved survival in bacteremia due to KPC-producing K. pneumoniae."
Article: Treatment Outcome of Bacteremia Due to KPC-Producing Klebsiella pneumoniae: Superiority of Combination Antimicrobial Regimens (http://aac.asm.org/content/56/4/2108.abstract)
Clavulanic Acid
Article: University of Notre Dame: Importance of position 170 in the inhibition of GES-type Beta-lacatmases by clavulanic acid (http://books.google.com/books?id=SKOcDOCZN_UC&pg=PA14&lpg=PA14&dq=potential+inhibitors+for+GES-5+carbapenemase&source=bl&ots=SZDECjfoPD&sig=YZtaTUXJ6Z0UoDU9URirksAd1Jw&hl=en&sa=X&ei=jDZgU4jPJMOGyASG7YHoAQ&ved=0CHMQ6AEwCQ#v=onepage&q=potential%20inhibitors%20for%20GES-5%20carbapenemase&f=false)
EC#: 3.5.2.6
Link to BRENDA EC# page: http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.2.6
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Penicillinase Benzylpenicillin as Substrate (commonly known as Penicillin G)
Method: Titrimetric
Cost: $17.00
Quantity: 356.37g
Supplier: BioReagent
CAS#: 69-57-8
Link for Procedure: http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/penicillbenzyl.pdf
Link for MSDS sheet: http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=P3032&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fsearch%3Finterface%3DAll%26term%3Dpenicillin%2Bg%26N%3D0%26mode%3Dmatch%2520partialmax%26focus%3Dproduct%26lang%3Den%26region%3DUS
Structure Available (PDB or Homology model)
PDB#: 4H8R
Pairwise alignment of your BLASTP search in NCBI against the PDB
Query Coverage: 26-129
Max % Identities: 33/104 (32%)
% Positives: 43/104 (41%)
Chain used for homology: Chain A
Current Inhibitors: dCHEMBL777, TAZOBACTUM, and CHEMBL403
Expression Information (has it been expressed in bacterial cells): Yes
Purification Method:
β-Lactamase purification.Cultures of E. coli TOP10 harboring recombinant plasmid pTOPO-GES-14 were grown overnight at 37°C in 4 liters of TS broth containing amoxicillin (50 μg/ml) and kanamycin (30 μg/ml). β-Lactamase GES-14 was purified by ion-exchange chromatography. Briefly, the bacterial suspension was pelleted, resuspended in 40 ml of 100 mM sodium phosphate buffer (pH 7.0), sonicated, cleared by ultracentrifugation, and treated with DNase. The extract was then dialyzed against 20 mM bis-Tris {[bis(2-hydroxyethyl-imino]tris(hydroxymethyl)methane}(pH 6.5) and loaded onto a preequilibrated Q-Sepharose column with the same buffer. The resulting enzyme extract was recovered in the flowthrough and dialyzed against 20 mM Tris-HCl (pH 7.5) overnight at 4°C. This extract was then loaded onto a preequilibrated (20 mM Tris-HCl [pH 7.5]) Q-Sepharose column, and the proteins were eluted with a linear NaCl gradient (0 to 0.5 M). The β-lactamase activity was eluted with NaCl at a concentration of 200 mM in the same Tris-HCl buffer. Finally, fractions containing the highest β-lactamase activities were pooled and subsequently dialyzed overnight against 100 mM phosphate buffer (pH 7.0). The β-lactamase activity was determined qualitatively using nitrocefin hydrolysis (Oxoid, Dardilly, France). The protein content was measured using the Bio-Rad DC protein assay.
Article: Carbapenem-Hydrolyzing GES-Type Extended-Spectrum β-Lactamase in Acinetobacter baumannii (http://aac.asm.org/content/55/1/349.long)
Image of protein (PyMol with features delineated and shown separately):
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MRFIHALLLAGIAHSAYASEKLTFKTDLEKLEREKAAQIGVAIVDPQGEIVAGHRMAQRFAMCSTFKFPLAALVFERIDSGTERGDRKLSYGPDMIVEWSPATERFLASGHMTVLEAA
QAAVQLSDNGATNLLLREIGGPAAMTQYFRKIGDSVSRLDRKEPEMSDNTPGDLRDTTTPIAMARTVAKVLYGGALTSTSTHTIERWLIGNQTGDATLRAGFPKDWVVGEKTGTCAN
GGRNDIGFFKAQERDYAVAVYTTAPKLSAVERDELVASVGQVITQLILSTDK
Length of your protein in Amino Acids: 287 residues
Molecular Weight of your protein: 31184.5 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
25565
Abs 0.1% (=1 g/l) 0.820, assuming all pairs of Cys residues form cystines
25440
Abs 0.1% (=1 g/l) 0.816, assuming all Cys residues are reduced
TMpred graph Image:
CDS Gene Sequence (paste as text only):
ATGTCACTGTATCGCCGTCTAGTTCTGCTGTCTTGTCTCTCATGGCCGCTGGCTGGCTTTTCTGCCACCGCGCTGACCAACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGGA
CTTTGGCGGCTCCATCGGTGTGTACGCGATGGATACCGGCTCAGGCGCAACTGTAAGTTACCGCGCTGAGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCTTTCTTGCTGCC
GCTGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGCTGGACACACCCATCCGTTACGGCAAAAATGCGCTGGTTCCGTGGTCACCCATCTCGGAAAAATATCTGACAACAGG
CATGACGGTGGCGGAGCTGTCCGCGGCCGCCGTGCAATACAGTGATAACGCCGCCGCCAATTTGTTGCTGAAGGAGTTGGGCGGCCCGGCCGGGCTGACGGCCTTCATGCGC
TCTATCGGCGATACCACGTTCCGTCTGGACCGCTGGGAGCTGGAGCTGAACTCCGCCATCCCAGGCGATGCGCGCGATACCTCATCGCCGCGCGCCGTGACGGAAAGCTTACA
AAAACTGACACTGGGCTCTGCACTGGCTGCGCCGCAGCGGCAGCAGTTTGTTGATTGGCTAAAGGGAAACACGACCGGCAACCACCGCATCCGCGCGGCGGTGCCGGCAGA
CTGGGCAGTCGGAGACAAAACCGGAACCTGCGGAGTGTATGGCACGGCAAATGACTATGCCGTCGTCTGGCCCACTGGGCGCGCACCTATTGTGTTGGCCGTCTACACCCGGG
CGCCTAACAAGGATGACAAGCACAGCGAGGCCGTCATCGCCGCTGCG GCTAGACTCGCGCTCGAGGGATTGGGCGTCAACGGGCAGTAA
GC% Content for gene: 61.5%
CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
Primer design results for 'tail' primers (this is just 2 sequences):